The formation of glucosylceramide, the predominant sphingolipid in plant tissues, was examined in microsomes from wax bean hypocotyls. Membranes were incubated with UDP-[14C]glucose in an assay mixture. The lipid extracts obtained from the assays were separated by thin-layer chromatography, and the radioactivity incorporated into glucosylceramide, steryl glucoside, and acylated steryl glucoside was determined. Although the formation of glucosylceramide was detected and characterized, several lines of evidence contradicted the assumption that UDP-glucose is the immediate glucose donor for glucosylceramide formation in plants: PDMP (DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), an inhibitor of ceramide glucosyltransferase in animal tissues, did not inhibit glucosylceramide formation in bean microsomes. Addition of UDP-glucose pyrophosphorylase during the assay to degrade UDP-[14C]glucose blocked the further production of labeled steryl glucoside, but did not prevent the continued formation of labeled glucosylceramide. Omitting UDP-[14C]glucose and including steryl [14C]glucoside in the assay resulted in the formation of labeled glucosylceramide. Collectively, these results suggest that glucosylceramide formation in plants does not utilize UDP-glucose as the immediate glucose donor, as has been demonstrated for the reaction in animal tissues, and that steryl glucoside serves as glucose donor for ceramide formation. This study, the first to examine glucosylceramide formation in plants, provides evidence for a novel enzymatic reaction in sphingolipid synthesis as well as a new, metabolic role for steryl glucoside in plant tissues.